1. Field
The present disclosure relates to compositions and methods for enhancing bone formation, increasing bone density, increasing interconnections of internal bone, increasing bone mass, treating cartilage and/or bone defects, or combinations thereof, in a subject in need thereof, improving maternal and/or child health during the stages of preconception, pregnancy, lactation, and/or postpartum, and increasing osteoblastic/osteogenic activity in mesenchymal stem cells (MSCs; also called “marrow stromal cells” or “multipotent stromal cells”). More particularly, said compositions comprise cocoa extract.
2. Description of Related Art
Theobromine (IUPAC name: 3,7-dimethyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione; also known as 3,7-dimethylxanthine) is a white (or colorless) bitter-tasting crystalline powder with a sublimation point of 290-295° C., a melting point of 357° C., and a molecular weight of 180.16 g/mol. The solubility of theobromine in water is 1.0 g/2 L; in boiling water, it is 1.0 g/150 mL, and in 95% ethanol it is 1.0 g/2.2 L. Theobromine is related chemically to caffeine and theophylline, and is found in numerous foods including chocolate, cocoa, tea leaves, and acai berries. The chemical structures of theobromine, theophylline (1,3-dimethylxanthine), and caffeine (1,3,7-trimethylxanthine) are given below as formulae I, II, and III, respectively.

Theobromine is found naturally in cacao beans (Theobroma cacao) at a concentration of from about 1.5% to about 3%, and in the husk of the bean at a concentration of from about 0.7% to about 1.2%, or about 15 to about 30 g/Kg (Winholdz, 1983). Though part of the same chemical family, one must distinguish the stimulant effects of theobromine from those of caffeine. Caffeine acts relatively quickly, and its main effect on humans is increased mental alertness; theobromine's effect is subtler, and causes a mood elevation that is milder longer-lasting than that of caffeine. Theobromine's plasma half-life (t1/2) in the bloodstream is six hours, while caffeine's is only two hours. Another difference is that theobromine is not physiologically addictive, producing no withdrawal symptoms after prolonged regular consumption, while caffeine has been proven to be physiologically addictive and linked with many cases of proven withdrawal.
As used herein, “theobromine” refers to theobromine, a salt or double salt of theobromine, or a co-crystal comprising theobromine.
Two independent studies conducted in the 1980's found that the average level of theobromine in eight varieties of commercial cocoa powder was 1.89% (Shively & Tarka, 1984 and Zoumas et al., 1980). Of particular relevance are the normal levels of theobromine found in commercially-available foodstuffs, shown below in TABLE 1.
TABLE 1Food TypeTheobromine Contenthot chocolate beverages65mg/5-oz servingchocolate milk (from instant or sweetened58mg/servingcocoa powder)hot cocoa (average of 9 commercial mixes)62mg/servingcocoa cereals*0.695mg/gchocolate bakery products*1.47mg/gchocolate toppings*1.95mg/gcocoa beverages*2.66mg/gchocolate ice cream*0.621mg/gchocolate milk*0.226mg/gchocolate pudding*74.8mg/servingcarob products*0-0.504mg/gSources: Zoumas, et al., 1980; Blauch & Tarka, 1983; Shivley & Tarka, 1984; Craig & Nguyen, 1984.*Theobromine content determined by HPLC/reverse-phase column chromatography
Dark chocolate contains the highest levels of theobromine per serving of any type of chocolate, but the concentrations tends to vary between about 0.36% and about 0.63%. To put this into perspective with the foodstuffs mentioned in TABLE 1, a one-ounce bar of dark chocolate contains about 130 mg of theobromine, while a one ounce bar of milk chocolate contains about 44 mg of theobromine. Thus, the concentration of theobromine in a one-ounce bar of dark chocolate is approximately two times the amount in a 5-ounce cup of hot chocolate. For a 143-pound human being to achieve a toxic level of theobromine in their blood, they would have to ingest approximately 86 one-ounce milk chocolate bars in one sitting.
Theobromine can also be isolated or produced as an amine salt (e.g., the ethylene diamine salt thereof) or a double salt thereof (e.g., with alkali metal salts or alkaline earth metal salts of organic acids, for example alkali or alkaline earth metal salts of acetic, gluconic, benzoic, or salicylic acid).
The double salts may be prepared either to make the theobromine more water soluble, or to make insoluble complexes.
In 1966, Strålfors reported a reduction of dental caries in hamsters that were fed diets rich in chocolate. The Strålfors study examined the effect on hamster caries by comparing cocoa powder, defatted cocoa powder, and cocoa fat. Pure cocoa powder inhibited dental caries by 84%, 75%, 60%, and 42% when the cocoa powder comprised 20%, 10%, 5%, and 2% percent of the hamster diet, respectively. Defatted cocoa showed a significantly higher anti-caries effect than did fat-containing cocoa powder, but cocoa butter alone (comprising 15% of the hamster's diet) increased dental caries significantly (Strålfors A. “Effect on Hamster Caries by Dialyzed, Detanned or Carbon-treated Water-Extract of Cocoa” Arch Oral Biol. 1966; 11:609-15.
In a follow up study, Strålfors studied the nonfat portions of the cocoa powder and demonstrated that cocoa powder washed with water possessed considerably less anti-cariogenic effect than unwashed cocoa powder. Nevertheless, Strålfors still observed a considerable anti-caries effect in the washed cocoa powder group, “indicating an existence of a non-water soluble cariostatic factor,” and alluded to the existence of “two caries-inhibitory substances in cocoa: one water-soluble, and another which is sparingly soluble in water” (Strålfors, A., 1966).
Subsequent studies suggested that apatite crystals grown in vitro in the presence of theobromine were significantly larger than those grown in the absence of theobromine (see, e.g., U.S. Pat. Nos. 5,919,426 and 6,183,711, each of which is incorporated by reference in its entirety). Ingestion of theobromine by lactating rats was correlated with increased hydroxylapatite crystallite size (higher crystallinity) in the whole first molars of nursing pups exposed to theobromine, versus controls, as well as increased resistance to acid dissolution (see id.). The femurs of nursing female pups exposed to theobromine demonstrated higher crystallinity, and were stronger and stiffer than gender-matched controls; the femurs of male pups, however, did not show this relationship (see id.).
The Hall-Petch relationship, however, dictates that the resistance of a solid material to permanent deformation (e.g., its indentation hardness) increases as the particle size decreases. Consequently, the increased hydroxylapatite crystallinity observed after exposure to theobromine—coupled with the Hall-Petch relationship—suggests that the resistance of bone and teeth to indentation and permanent deformation should decrease after exposure to theobromine due to the larger crystal size. This suggestion finds support in recent work, demonstrating that “the hardness of [hydroxylapatite] follows the Hall-Petch relationship as the grain size decreases from sub-micrometers to nanometers” (Wang J. et al “Nanocrystalline hydroxyapatite with simultaneous enhancements in hardness and toughness” Biomaterials. 2009; 30:6565-72). Another study suggests that the “hardness” of hydroxylapatite has little to do with particle size, showing almost no change in hardness with decreasing grain size, yet demonstrates that the “fracture toughness” of hydroxylapatite is increased with decreasing particle size (Mazaheri M, et al “Effect of a novel sintering process on mechanical properties of hydroxyapatite ceramics.” J. Alloys Compd. 2009; 471:180-4). A study of human adult and primary (baby) teeth demonstrated that, “[w]hen compared to the adult tooth, the baby enamel was thinner, softer, more prone to fracture, and possessed larger [hydroxylapatite] grains” (Low I M, et al. “Mapping the structure, composition and mechanical properties of human teeth.” Mater Sci Eng C Mater Biol App. 2008; 28:243-47.).
Because of such conflicting and paradoxical results, one cannot extrapolate the known characteristics and responses of hydroxylapatite to environmental factors to predict a reliable or accurate result cannot be predicted simply by evaluating the prior art and extrapolating a result.
MSCs are characterized by their ability to produce daughter stem cells, and to differentiate into many distinct cell types including, but not limited to, osteoblasts, stromal cells that support hematopoiesis and osteoclastogenesis, chondrocytes, myocytes, adipocytes of the bone marrow, neuronal cells, and β-pancreatic islet cells. Consequently, MSCs provide osteoblasts and stromal cells needed for bone development, bone remodeling and hematopoiesis throughout life.
In humans, bone formation begins during the first 6-8 weeks of fetal development. Progenitor stem cells of mesenchymal origin migrate to predetermined sites, where they either: (a) condense, proliferate, and differentiate into bone-forming cells (osteoblasts), a process observed in the skull and referred to as “intramembranous bone formation;” or, (b) condense, proliferate and differentiate into cartilage-forming cells (chondroblasts) as intermediates, which are subsequently replaced with bone-forming cells. More specifically, mesenchymal stem cells differentiate into chondrocytes. The chondrocytes then become calcified, undergo hypertrophy and are replaced by newly formed bone made by differentiated osteoblasts, which now are present at the site. Subsequently, the mineralized bone is extensively remodeled, thereafter becoming occupied by an ossicle filled with functional bone-marrow elements. This process is observed in long bones and referred to as “endochondral bone formation.” In postfetal life, bone has the capacity to repair itself upon injury by mimicking the cellular process of embryonic endochondral bone development. That is, mesenchymal progenitor stem cells from the bone-marrow, periosteum, and muscle can be induced to migrate to the defect site and begin the cascade of events described above. There, they accumulate, proliferate, and differentiate into cartilage, which is subsequently replaced with newly formed bone.
MSCs are extremely rare in the bone marrow, and earlier attempts to expand them ex vivo from rodent or human marrow have proven difficult. Moreover, inducing proliferation and differentiation of progenitor cells into functional bone, cartilage, tendon, and/or ligamentous tissue has been shown to require various proteins, including, but not limited to, members of the family of bone morphogenetic proteins (BMPs) and members of the TGF-β superfamily of growth factors. Manufacture, isolation, and purification of such proteins is necessarily more complex and expensive than that of small-molecule chemical compounds because proteins are larger, more complex, and are synthesized by a living cell.
Thus, there remains a need for small-molecule chemical compounds—especially ones known to be non-toxic—capable of inducing proliferation of progenitor cells into functional bone, cartilage, tendon, and/or ligamentous tissue.
The solution to this technical problem is provided by the embodiments characterized in the claims.